The experimental goals described in this new FIRCA application address fundamental molecular mechanisms underlying the cause of vascular endothelial cell dysfunction and degeneration observed in the course of hyperglycemia and the vascular complications of diabetes. They also serve as the foundation for further expanding the mutually beneficial collaborative research and training activities in free radical biology and medicine shared by investigators at the University of Alabama at Birmingham School of Medicine and the Facultad de Medicina of the Universidad de La Republica in Montevideo, Uruguay. Specifically, the theme of the parent grant "Nitric Oxide-Dependent Oxidative Lung Injury" addressing the hypothesis that pulmonary exposure to endogenous and exogenous NO, in the face of oxidative stress, leads to tissue oxidative damage, will be expanded in a new and important direction by addressing the novel concept that during hyperglycemia enhanced rates of mitochondrial-derived superoxide (O2) react with NO, decreasing its cell signaling functions and promoting the formation of the cytotoxic and pro-apoptotic reactive intermediate peroxynitrite anion (ONOO). This hypothesis provides an unifying mechanism accounting for the decreased bioavailability of NO during hyperglycemia, thus impairing endothelial-dependent vasodilation, as well as the degenerative and thrombogenic processes observed at diabetic endothelium, postulated to be triggered by ONOO-mediated modification and release of pro-apoptotic mitochondrial components such as cytochrome c. To test this hypothesis, a series of studies ranging from biochemical to cellular level will be enacted by pursuing the following Specific Aims: 1) Explore the impact of nitric oxide reaction with mitochondrial superoxide on nitric oxide bioavailability and apoptotic signaling, 2) Evaluate the role of nitric oxide-derived oxidants in hyperglycemic injury to vascular endothelial cells. Thus, these specific aims extend a theme central to our research endeavors namely, that NO signaling is potently modified by oxidative stress and often yields pathogenic secondary mediators. In this proposal, we address for the first time the interplay between mitochondrial-derived O2 and NO, which we view to be critical during the development of the vascular cell dysfunction and injury observed in diabetes, and from this new insight will reveal new perspectives for unique pharmacological approaches to treat diabetic vasculopathy.